PROJECT SUMMARY The KRAS oncogene is mutated in ~95% of pancreatic ductal adenocarcinoma (PDAC). There is considerable experimental evidence that continued expression of mutant KRAS is essential for PDAC maintenance. It is generally accepted that an effective anti-KRas therapy will have a significant impact on pancreatic cancer, with inhibition of KRAS effector signaling considered the most promising direction for advancement to the clinic. In particular, considerable effort and interest is now focused on inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase (MAPK) cascade. However, Raf and MEK inhibitors have shown limited to no efficacy in RAS- mutant cancers, due primarily to cancer cell adaptation and ERK reactivation to overcome the inhibitor action. These findings have prompted the development of ERK inhibitors, with four inhibitors recently entering clinical evaluation. Among them, BVD-523, a small molecule that targets ERK1 and ERK2 in the sub-nanomolar range is the leading compound entering oncology clinical trials (NCT01781429). In our revised proposal, we now provide substantial preclinical and clinical analyses of BVD-523 that support the rationale and feasibility of our studies. The innovation of our studies is our focus on a first-in-class direct inhibitor of ERK and applying unbiased genetic and chemical library screens to identify combination therapies to overcome limitations for its use for PDAC. We also address the concern raised in our previous submission regarding undefined clinical studies. We propose four Specific Aims to advance the clinical development of BVD-523 for PDAC treatment. We will: (1) clinically evaluate BVD-523 anti-tumor activity and biomarkers of response in patients with PDAC; (2) identify molecular mechanisms for acquired resistance to BVD-523 in KRAS-mutant PDAC; (3) identify combination inhibitor approaches that overcome de novo resistance and render BVD-523 treatment cytotoxic; and (4) assess combination inhibitor strategies with BVD-523 for anti-tumor activity in state-of-the-art organoid culture and mouse models of pancreatic cancer. Our goal is to identify combinations that overcome de novo and acquired resistance, as well as cytostatic and transient responses and normal tissue toxicity, for future clinical evaluation. When completed, our study will have identified predictive biomarkers for ERK treatment response, allowing us to identify the most effective ERK combinations to be tested in clinical studies.